PROJECT SUMMARY Our objective is to develop a small molecule therapy for the treatment of heart disease associated with Duchenne Muscular Dystrophy (DMD) - a disease that occurs in 1 out of every 3500 male births, and for which there is no cure or long-term effective treatment. Heart failure (HF) is a contributing cause of mortality among persons afflicted with DMD. A key abnormality in HF is defective handling of calcium that has been partially related to abnormal sarcoplasmic reticulum (SR) function in cardiac myocytes. Reduced expression and altered activity of the cardiac SR Ca2+-ATPase (SERCA2a) have been found in human and animal models of HF. Our group has described a role for the small ubiquitin-like modifier type 1 (SUMO1) as a regulator of SERCA2a and has shown that gene transfer of SUMO1 in rodents and large animal models of HF restores cardiac function. Through an extensive small molecule screen, we have identified and characterized a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identified a unique binding pocket on SUMO E1 that is responsible for N106?s effects. We have completed pharmacokinetics, toxicity, and off-target studies on this compound along with detailed biological activities in vivo. In this proposal, we provide preliminary evidence that SUMO1 pathway was impaired in mouse DMD hearts, which was accompanied by SERCA2a dysfunction, and for the feasibility of our novel therapy in DMD. The key objective of this Phase I SBIR proposal is to identify candidate hits small-molecule activators of SERCA2a SUMOylation from a set of new scaffolds. We have used computational modeling to perform in silico screening of the validated E1 enzyme pocket to identify new scaffolds for further evaluation. In our first aim we propose synthesizing, and testing the efficacy and ADME/PK of our novel chemical classes of small molecule activators (i.e. 5 different new scaffolds) to tailor an improved PK profile than N106 and increased half-life. The SAR study, together with the SUMO E1 activity assay, will identify and prioritize hits-to-leads. In our second aim, we will determine the potency, the safety, and the molecular mechanism of action of our three selected lead candidates. A combination of SERCA2a SUMOylation assays and cardiomyocytes experiments will define mechanisms of action, and together with the PK studies will identify lead compounds for future development. Finally, we will test the efficacy of our lead therapeutic in a mouse model of DMD. Testing oral doses of small molecule activators of SUMOylation in established DMD models will allow us to show improvement in cardiac function and cardiac pathology. The completion of these studies will guide us towards a path for future clinical application in Phase II SBIR plan.